The trachea and esophagus (TE) arise from a single foregut tube in early fetal development. Defects in TE morphogenesisresultinaspectrumoflife-threateningcongenitaltracheo-esophagealbirthdefects(TEDs)that prevent proper breathing or feeding in newborns. The goal of this project is to determine the molecular and cellular basis of TEDs using animal models. Corrected surgically in the neonatal period, TEDs are often associated with long-term co-morbidity. Occurring in ~1:3500 births, the etiology of TEDs is poorly understood.Althoughevidenceindicatesamajorgeneticcomponent,knownmutationsin14genesaccountfor only12%ofpatientswithesophagealatresiaand/ortracheoesophagealfistula(EA/TEF)[1],whilethegenetic basis of more rare and lethal tracheal atresia (TA) is unknown. Sporadic mutations in ~25 additional genes have been associated with EA/TEF patients, but these remain to be validated. Mouse has proven to be effectiveformodelingTEDs,andindicatesakeyrolefortheHedgehog(HH)andBMPpathways,withmutants exhibitingdefectssimilartohumanpatient.Despitethisprogressthereareanumberoflimitationsinthefield. Mouse is a relatively low throughput model and only a few of candidate mutations from patients have been validatedtodate.Second,whileHHandBMPareimplicatedTEmorphogenesisthecellularmechanismsthey regulate, to control separation of the foregut tube into esophagus and trachea are unknown. This is in part because these events occur early in fetal development when internally developing mouse embryos are challenging to manipulate and visualize. In preliminary data we have established Xenopus embryos as an innovative high-throughput model to complement mouse genetics, and have begun to identify novel and conservedcellularmechanismscontrollingTEmorphogenesis.ThesestudiesleadustohypothesizethatHH andBMPinteracttoregulatethecellularprocessesofTEmorphogenesisandthatmutationsinthese pathways result in a spectrum of phenotypes that model human TEDs. This project will define the molecular and cellular mechanisms of TE development, define the structural basis of TEDs and test putative TED-causingmutationsfrompatients(project-1).Ultimatelythiswillimprovediagnosis,enhancepatientcare, andinformstrategiestogenerateTEtissuefromhumanpluripotentstemcells(hPSCs)(project-3). Aim1CharacterizethecellularmechanismsofTEmorphogenesisinanimals. Aim2DeterminehowdefectsinHH-GliandBMP-Sox2pathwaysdisruptTEmorphogenesis. Aim3ValidatecandidateTED-causingmutationsinXenopusandmouse.